Genomic DNA Extraction and RNA-Seq Analysis

Whole-cell genomic DNA was isolated from either the JRB310 wild-type strain, the wild-type sexual progeny (exconjugants) or dcl-1 mutants, dcl-1²³ and dcl-1⁴¹, and rdrp mutants, rdrp¹³, rdrp¹⁷, and rdrp²⁵ using the Nucleospin Tissue DNA Extraction kit (Macherey-Nagel). One microgram of total genomic DNA was used to prepare Illumina libraries using a standard manufacturer's protocol and run on Illumina HiSeq 2500. Small RNAs were gel-purified from total RNA and loaded on denaturing 7 M Urea, 15% polyacrylamide gel. Of note, 17- to 25-nt small RNAs or 30- to 50-nt RNA (from dcl-1⁴¹) were gel purified and cloned using the Illumina Truseq small RNA library construction kit. For RNA-seq, total RNA was isolated from wild-type reference strain JRB310 and two mutant strains, dcl-1⁴¹ and rdrp¹³. Poly(A) RNA enrichment was performed using the polyA Spin mRNA Isolation Kit (NEB). Enriched RNA was reverse transcribed using Superscript III (ThermoFisher) and strand-specific libraries were constructed using published methods (Zhang et al. 2012 (link)). Barcoded libraries were mixed and sequenced.